title: Identification of folding intermediates of streblin, the most stable serine protease: biophysical analysis.
abstract: Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, reeteshmbu@gmail.com.
authors.forename: Reetesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Pinki
authors.initials: P
authors.lastname: Tripathi
authors.forename: Fabio Rogerio
authors.initials: FR
authors.lastname: de Moraes
authors.forename: Icaro P
authors.initials: IP
authors.lastname: Caruso
authors.forename: Medicherla V
authors.initials: MV
authors.lastname: Jagannadham
mesh_terms: D055592:Biophysical Phenomena / Q000187:drug effects; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D057057:Serine Proteases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D057057:Serine Proteases; D019791:Guanidine